The role of a sodium ion binding site in the allosteric modulation of the A(2A) adenosine G protein-coupled receptor

Abstract

The function of G protein-coupled receptors (GPCRs) can be modulated by a number of endogenous allosteric molecules. In this study, we used molecular dynamics, radioligand binding, and thermostability experiments to elucidate the role of the recently discovered sodium ion binding site in the allosteric modulation of the human A(2A) adenosine receptor, conserved among class A GPCRs. While the binding of antagonists and sodium ions to the receptor was noncompetitive in nature, the binding of agonists and sodium ions appears to require mutually exclusive conformational states of the receptor. Amiloride analogs can also bind to the sodium binding pocket, showing distinct patterns of agonist and antagonist modulation. These findings suggest that physiological concentrations of sodium ions affect functionally relevant conformational states of GPCRs and can help to design novel synthetic allosteric modulators or bitopic ligands exploiting the sodium ion binding pocket.

Figure 1

Structure and conservation of the central sodium ion-binding allosteric pocket in Class A GPCRs. (A) The Na⁺ distorted octahedral coordination as in the A_2AAR crystal structure: The first shell is occupied by two conserved polar residues (green) and three water molecules, which contact with a second shell of residues (cyan), or with a second layer of water molecules connecting with the third shell of residues (magenta). (B) Sequence conservation of the 15 residues lining the binding pocket among inactive GPCR crystal structures. (C) Structure of the A_2AAR complex with ZM241385, showing residues with higher than 50% conservation in all Class A receptors as sticks with green carbons. (D) A close-up of the central allosteric pocket (transparent blue surface), showing the side chains located within 5 Å from the ten waters of the sodium ion-water cluster (green sticks: A_2AAR; gray thin lines: the corresponding side chains of the overlaid GPCR crystal structures depicted in panel B). See also Supplementary Figure S1.

Figure 2

The two coordination states of the sodium ion as observed in MD simulations. (A) Volumetric density map (isosurface contoured at 0.3 Å⁻³ value, blue) corresponding to the sodium occupancy as calculated from simulations MDS1 (see Table 1). The starting crystal structure is displayed, together with the electron density (contoured at 1 σ level, black). (B) The time-evolution of the distances between the sodium ion and Asp^2.50 (blue), Ser^3.39 (red) and Asn^7.45 (green), shown for the 3 independent replicates (R1–R3) of MDS1. The corresponding distances are denoted as dashed lines in panel (A) with the same color code, and are the source of the data in Table 2. The RMSD of the ion with respect to its crystallographic position is indicated with a black line, while the horizontal bar at 1.8 Å (the resolution of the parent crystal structure) denotes the limit for the crystallographic coordination state (position c1). See also Table S1 and Figure S2.

Figure 3

Rotameric transitions of Trp^6.48 and Asn^7.45 in the apo simulations of the inactive A_2AAR. (A) Populations of the initial conformational states in the simulations with (MDS2) and without (MDS4) the sodium ion, and the number of waters in the ion binding site (each data is an average of the 3 MD replicas). Note that Trp^6.48 only finds the trans conformation when not in the initial g+ conformation, while Asn^7.45 is more flexible and can be found in either trans, g+ or the initial g− conformations. (B) Representative snapshot (magenta) of the conformation of these two residues in MDS2 and (C) MDS4, with the reference crystal structure overlaid in light gray.

Figure 4

The movement of helix VII (orange) in order to accommodate the sodium ion. (A) Starting (grey) and ending (rainbow) conformations of the agonist-bound A_2AAR in the presence of sodium ion (MDS5), with the inactive crystal structure denoted in anthracite. (B) The distance between helices III and VII (X axis, Cα of residues Ile^3.40 and Asn^7.45 as indicated by a dashed line in the 3D-structure) is plotted against the backbone RMSD of the motif His^7.43-Asn^7.49, (Y axis), using as a reference the active conformation of A_2AAR. Each dot is a snapshot extracted every 0.5 ns, with the time evolution depicted by the shading code (light grey -> black). Starting and ending conformations indicated with an asterisk and a triangle, respectively.

Figure 5

Impact of amiloride and HMA in the binding of antagonists. (A) Amiloride docking (magenta carbons) induces a shifted position of Trp246^6.48 side chain, revealing potential steric clashes with the orthosteric ligand ZM241385 (yellow carbons, superimposed from the crystal structure with PDB code 4EIY). (B) Same conformation of the amiloride- bound A_2AAR, with the caffeine pose (green carbons) superimposed from the crystal structure of A_2AAR/caffeine complex (PDB code 3RFM). (C) Flexible docking of HMA (magenta carbons) is predicted to further shift Trp246^6.48 and interfere with ZM241385 binding. (D) Mobility of the antagonists in the presence or absence of amiloride, HMA and sodium in the allosteric pocket, calculated as the RMSF from the MD simulations (dark shaded bars for ZM241385; light shaded bars for caffeine). The error bars indicate the standard deviation estimated from three MD replicas (n=3); ami=amiloride; ZM= ZM241385. Significantly different from the control simulation (i.e. absence of any orthosteric ligand) in a student t-test with *p < 0.05.

Figure 6

Equilibrium displacement of [³H]ZM241385 and [³H]NECA by allosteric modulators. (A) NaCl, (B) amiloride, and (C) HMA. Experiments performed in duplicate on human A_2AARs transiently expressed in HEK293T cell membranes. Associated IC₅₀ values listed in Table S2.

Figure 7

Effect of allosteric binders on A_2AAR thermostability measured by CPM assays. (A) Titration of NaCl effect on A_2AAR thermostability, mean ± S.E.M. shown for measurements performed in triplicate. (B) Effect of NaCl (150 mM), amiloride (100 μM), caffeine (500 μM), ZM241385 (50 μM), UK432097 (50 μM) and combinations thereof on A_2AAR thermostability.